Previously, we have reported that the active manganese form of glutamine synthetase (GS) undergoes a reversible, thermally induced partial unfolding without dissociation of the dodecamer (622,000 MW). The transition (T = 51.6 degrees C; deltaH about 0.3 cal/g) involves a coupled, cooperative partial unfolding of two domains in each of the twelve subunits. However, at T > 68 degrees C, GS precipitates in the presence of 1 mM Mn(II) and 100 mM KCl at pH 7. Recent studies have shown that removal of KCl results in a destabilization of the reversible transition (T about 46 degrees C) and the appearance of a second much larger endotherm (T about 81 degrees C; deltaH about 5.6 cal/g), which is irreversible, scan-rate dependent, and accompanied by aggregation. The latter transition results in about 50% disruption of secondary structures. Aggregation is inhibited by the addition of 3 M urea in DSC experiments and the dodecamer completely dissociates to monomers. In the presence of 3 M urea, the unfolding transition occurs at T approximately 62 degrees C with an apparent deltaH approximately 4 cal/g. Previously, titration calorimetry for urea addition to GS has yielded a reliable value of deltaH = 14 plus/minus 4 cal/g for complete dissociation and unfolding of GS (Zolkiewski, M., Nosworthy, N. J., and Ginsburg, A., Protein Science 4, 1544-1552, 1995). The same studies have shown that urea binds to multiple, independent sites of Mn-GS with the number of urea binding sites increasing about 9-fold during GS unfolding. Thus, the lower endothermic enthalpy observed in thermal unfolding studies of Mn-GS can be attributed to thermostable structural regions and aggregation (in the absence of urea), and exothermic binding of urea to the unfolded monomer in the presence of urea (about-10 cal/g). HPLC-gel filtration studies show only folded dodecamer and unfolded monomer during the thermal transition, which suggests that thermodynamically stable intermediate species are absent during the thermally induced dissociation and unfolding of GS. A 10-fold dilution of the stable monomer formed at 65 degrees C produces a slow regeneration of about 50% enzymatic activity at 20 degrees C.
